1. Field of Invention
The present invention relates to methods and compositions for the time released delivery of triaqua-mu3-oxohexakis-mu-alkylcarboxylatotrichromium (1+), [Cr3O(carboxylate)6 (H2O)3]+.
2. Discussion of the Background
In the late 1950s and 1960s, rats fed a chromium-deficient diet were found to possess a decreased ability to repress blood glucose concentrations, while chromic ions were shown to increase the efficiency of insulin action in rat epididymal tissue [1-5]. Since these observations, a search has been underway to identify the biologically active form of chromium, that is, the biomolecule which naturally binds chromium (III) and possesses an intrinsic function associated with insulin action in mammals [6-8]. The average American diet contains only about 30 μg Cr per day [9, 10], which has resulted in the development of chromium-containing dietary supplements. Such materials also have potential as insulin-potentiating therapeutics which could possibly see use in the treatment of diabetes and related conditions [11]. Determining the structure, function, and mode of action of the biologically active form of chromium could greatly aid in the rational design of such potential therapeutics.
The first chromium-containing species proposed to be biologically active was glucose tolerance factor(GTF)[1,12]. GTF was first isolated from acid-hydrolyzed porcine kidney powder, although a similar, if not identical, material was subsequently isolated from yeast[1,13]. Currently the term GTF is usually understood to refer to only the material isolated from yeast. GTF is absorbed better than simple chromic salts and potentiates insulin action in rat epididymal tissue or isolated rat adipocytes [14]. However, kinetics studies indicate that GTF does not intrinsically possess biological activity [15]; additionally, the material is apparently a byproduct of the acid hydrolysis step used in its purification [16].
GTF was proposed to be composed of chromic ion, nicotinic acid, and the amino acids glycine, glutamic acid and cysteine [13]. While these results have not been reproducible in some laboratories [17-21], this report stimulated an intense interest in the synthesis of chromic-nicotinate complexes [22-25], some of which have been patented as nutritional supplements. The proposed identification of nicotinic acid (2-carboxypyridine) also stimulated investigations of complexes of chromium(III) with the related pyridine carboxylic acids picolinic acid (2-carboxypyridine) and isonicotinic acid (4-carboxypyridine) [26-28]. As a result chromium(III) tris(picolinate), Cr(pic)3, has become a very popular nutritional supplement and is being tested as a therapeutic for the treatment of symptoms of adult-onset diabetes. It is available over-the-counter in the form of pills, chewing gums, sport drinks, and nutrition bars. Cr(pic)3 is also a well absorbed form of chromium and has been proposed to be the biologically active form of chromium [29]. This is, however, extremely doubtful given the chemistry required to synthesize this material.
In the last decade, a number of investigators have examined the effects of administering Cr(pic)3 (and in some cases other forms of chromium(III)) to rats on regular diets [30-33]. After an initial preliminary report which suggested beneficial effects on blood variables [30], detailed examinations of the effect of Cr(pic)3 administration in amounts up to 1500 μg/kg diet for up to 24 weeks have found no acute toxic effects [31-33]. However, the compound and other chromium sources examined (most notably “Cr nicotinate” and chromium chloride) also had no effect on body mass, percentage lean or fat content, tissue size (heart, testes, liver, kidney, muscle, epididymal fat, spleen, and kidney), or blood variables (fasting glucose, insulin, cholesterol, etc.). No differences in the gross histology of the liver or kidney (organs where chromium(III) preferentially accumulated) were found, although chromium did accumulate in these organs [33]. Another study compared the effects of a Cr-deficient diet with diets supplemented with ten different sources of chromium, including allowing rats to live in stainless steel cages. The Cr sources had no effect on body mass; all but one source decreased epididymal fat. Testes and liver masses tended to be lowered, whereas kidney, heart, and spleen masses were not significantly altered. Supplemental Cr had no effect on serum triglycerides or cholesterol, and only one source resulted in lower serum glucose [34]. While these studies did not manifest any acute toxicity, the lack of beneficial effects of Cr(pic)3 supplementation on growth, fat content or glucose, insulin, or cholesterol concentrations raises questions about its therapeutic potential. Recently the safety of intaking Cr(pic)3 has been questioned, especially in regards to its potential to cause clastogenic damage [35,36]. At physiologically-relevant concentrations of chromium (120 nM) and biological reductants such as ascorbic acid and thiols (5 mM), Cr(pic)3 has been shown to catalytically produce hydroxyl radicals which cleave DNA[35]. This ability stems from the combination of chromium and picolinate; the picolinate ligands prime the redox potential of the chromic center such that it is susceptible to reduction. The reduced chromium species interacts with dioxygen to produce reduced oxygen species including hydroxyl radical. These studies are in agreement with earlier studies which showed that mutagenic forms of chromium(III) required chelating ligands containing pyridine-type nitrogens coordinated to the metal [37].
Recently the naturally-occurring oligopeptide low-molecular-weight chromium-binding substance, LMWCr, has been proposed as a candidate for the biologically active form of chromium [6,7,38,39]. Kinetics studies of insulin action on rat adipocytes suggest that LMWCr has an intrinsic function in insulin-sensitive cells [15,40]. The oligopeptide appears to be part of an insulin signal amplification mechanism [6,7]. The oligopeptide containing four chromic ions binds to insulin-activated insulin receptor, stimulating its tyrosine kinase activity up to eight-fold with a dissociation constant of approximately 100 pM [38]. Spectroscopic studies have shown that LMWCr possesses a multinuclear chromic assembly where the chromic centers are bridged by anionic ligands (presumably oxide and/or hydroxide). The assembly is supported by carboxylate groups from aspartate and glutamate residues from the oligopeptide [41]. This discovery has spurred an interest in the synthesis and characterization of multinuclear oxo(hydroxo)-bridged chromium(III) carboxylate assembles [42-45]. In 1997, such an assembly, [[Cr3O(O2CCH2CH3)6(H2O)3]+, 1, was found to mimic the ability of LMWCr to stimulate insulin receptor kinase activity [39]. Both LMWCr and the biomimetic 1 have been proposed as potential nutritional supplements and therapeutics. Both LMWCr and 1 have been shown not to lead to DNA cleavage [46]. The synthetic complex has several potential benefits over the natural material: it is inexpensive to synthesize and can be readily prepared in bulk. LMWCr is susceptible to hydrolysis, especially in the presence of acid, whereas the synthetic material can be recrystallized from dilute mineral acid [47] and could potentially survive oral ingestion. After the insulin signaling event, LMWCr may be excreted in the urine [48,49], and it is possible the body might target the material for excretion rather than absorption.
The biologically-active, naturally-occurring oligopeptide low-molecular-weight chromium-binding substance (LMWCr) has been found to activate the insulin-dependent tyrosine protein kinase activity of insulin receptor (IR) approximately eightfold with a dissociation constant of circa 250 pM. [38] This activity is directly proportional to the Cr content of the oligopeptide (being maximal at four chromic ions per oligopeptide), while substitution of chromium with metal ions commonly associated with biological systems results in inactivating the oligopeptide. Similarly, LMWCr has been reported to activate a membrane-associated phosphotyrosine phosphatase; this activation also requires four chromic ions per oligopeptide to be maximal, while chromic ions could not functionally be replaced with other transition metal ions. [50] A role for LMWCr in amplification of insulin-signaling has been postulated. [38,41]. Chromium is mobilized from the blood and taken up by insulin-dependent cells in response to insulin. [51] LMWCr is maintained in its apo form [52] but possesses a large chromic ion binding constants(s) as it is capable of removing chromium from Cr-transferring. [52,53] The holo LMWCr is then capable of stimulating IR kinase activity, amplifying the signal of insulin into the insulin-dependent cells. An association between chromium and insulin-dependent glucose and lipid metabolism has been reported for nearly four decades; [54] however, only recently since procedures for isolation of quantities of LMWCr suitable for kinetic and spectroscopic studies have been developed. [41] has progress been made in understanding the association on a molecular level.
An association between the essential nutrient chromium and adult-onset diabetes has also been postulated. [55] Anderson and coworkers found improved glycemic control for 180 adult-onset diabetic patients following chromium supplementation, [56] while Ravina and Slezack using 138 adult-onset diabetic patients found reduced insulin requirements. [57] Unfortunately, the form of chromium used as a dietary supplement in these studies, chromium(III) picolinate, has been found to cause chromosome damage. [58] This suggests that a new form of chromium for use as a dietary supplement and as part of a potential treatment for adult-onset diabetes is required.
LMWCr would appear to be a possibility. It has a high LD50[53] and is biologically active, opposed to chromium picolinate and glucose tolerance factor (a material isolated from acid-hydrolyzed Brewer's yeast extracts) which serve only as sources of readily absorbable chromium. [59] However, LMWCr is susceptible to hydrolysis under acidic conditions[16] and consequently could not be taken orally without degradation.
Recent studies have shown that biomimetic 1 is absorbed with 40-60% efficiency over a wide range of dosage, a significant improvement over the 0.5-2% absorption of other chromium supplements such as chromium chloride, chromium nicotinate, and chromium picolinate. [60] However, 1 is absorbed and enters tissues very quickly such that methods to maintain levels of 1 would be of value. Accordingly, there remains a need for improved dietary supplements.